In most assisted reproduction programs, gonadotropins are used alone or in combination to stimulate the growth and maturation of multiple oocytes. This is essential because of the need to recruit a greater number of follicles, which improves the chance of fertilization and an increased number of embryos for transfer to give acceptable success rates. Recent advances in the understanding of ovarian stimulation, the techniques of oocyte retrieval, the handling of gametes, the methods of assisted fertilization and improved conditions of culture media have steadily increased the fertilization rate. Oocyte fertilization rates of 60-70% can now be expected when conventional insemination or intracytoplasmic sperm injection (ICSI) are carried out. However, there has not been a corresponding increase in implantation rates, which have remained steady at 10-15% per embryo1.
It is believed that the high, supraphysiologic levels of estrogen, attained during ovarian stimulation, may result in an adverse effect of ovarian stimulation on the outcome of infertility treatment. Significant decreases in pregnancy and implantation rates have been observed when estradiol concentrations were >10,000 pmol/L compared with patients having lower estradiol concentrations2. High serum estradiol concentrations on the day of HCG injection in IVF patients, regardless of the number of oocytes retrieved, were found to be detrimental to uterine receptivity3. Recently, it has been shown that a significant reduction in implantation and pregnancy rates occurred in almost all women with a serum estradiol concentration greater than 20,000 pmol/L4.
Different mechanisms have been postulated to explain the adverse effect of the supraphysiologic levels of estrogen including deleterious effects on the endometrium and the embryo, although the exact mechanisms have not yet been determined.
Effect of Supraphysiologic Levels of Estrogen on Implantation:
There is controversy regarding the effect of ovarian hyperstimulation on endometrial development. Most investigators have reported adverse effects of high estrogen levels on endometrial development but there was no consensus on the actual mechanism of this effect. Endometrial biopsies for dating have shown both endometrial advancement and endometrial retardation in relation to high serum estradiol concentrations. However, all studies confirm direct effects on endometrial development that may jeopardize the chance of implantation due to the lack of synchronization between the endometrium and early embryo development. Such synchronization is crucial for successful implantation (window of implantation).
Various studies have shown a high incidence of endometrial glandular advancement5 and retardation6 using morphological and immunohistochemical criteria7. One study of natural versus ovarian stimulation cycles, demonstrated an advanced development of the ultrastructure of endometrial surface epithelium in the stimulation cycles8. However, another investigation demonstrated that ovulation induction was not associated with abnormal endometrial development9.
More recently, the effect of excessively high estradiol concentrations (>20,000 pmol/L) were found to be associated with a deficient secretory transformation of the endometrium and a suboptimal endometrial environment for implantation. This finding supports clinical observations of significantly lower pregnancy rates in IVF cycles in women with estradiol concentrations ≧20,000 pmol/l. In these patients, there was a marked stromal oedema associated with a significantly greater number of vessels, and advanced stromal maturation possibly representing a direct effect of high estradiol levels on the endometrium10. In another study, an asynchronous development of endometrial glands and stromal was found in women undergoing IVF11.
Most recently, a detrimental effect of high estrogen concentrations has been demonstrated on the embryo itself. Decreased blastocyst formation and reduce embryo adhesion to an endometrial cell layer was observed in the presence of elevated estrogen concentrations12.
It appears that excessive estradiol production during controlled ovarian stimulation leads to insufficient secretory transformation of the endometrium and a discordant glandular and stromal development at a time that coincides with the period of maximum uterine receptivity. In addition, there are possible adverse effects directly oh the embryo that could reduce the chance for implantation. This may explain the findings of decreased implantation and pregnancy rates in IVF when serum estradiol concentrations are exceptionally high.
Measures to Improve Pregnancy Outcome by Reducing E2 Levels:
Different approaches have been suggested to improve the treatment outcome during assisted reproduction by reducing the intensity of ovarian stimulation to reduce the high estrogen levels. These approaches included minimal stimulation IVF cycles and natural cycle IVF which have been reported to be effective methods of treatment for ovulatory women undergoing assisted conception13. Other measures to lower estrogen levels include decreasing the FSH dose (step down protocol). With the use of a step-down FSH regimen in high responders, uterine receptivity may be improved secondary to lowering E2 levels during the preimplantation period14. Coasting or withholding FSH injections for a period of time prior to administration of hCG has been suggested in patients at substantial risk for the development of severe ovarian hyperstimulation syndrome (OHSS) and is associated with lowered estradiol levels15. However, all these measures are associated with the major drawback of decreasing the number of oocytes retrieved and embryos produced.
Aromatase Inhibition
As discussed above, the undesirable effects of ovarian stimulation on the outcome of infertility treatment may be due to the supraphysiologic levels of estrogen. Lowering estrogen levels may be associated with improved outcome by improving the implantation and pregnancy rates in addition to lowering risk of severe ovarian hyperstimulation syndrome. Reducing estrogen synthesis by aromatase inhibition during assisted reproductive technologies could be a way to ameliorate the deleterious effects of the supraphysiologic levels of estrogen during ovarian stimulation.
Until recently there was no effective aromatase inhibitor that could be used clinically to reduce estrogen levels during ovarian stimulation. This is because the available aromatase inhibitors (e.g. aminoglutethemide) lacked specificity to inhibit the aromatase enzyme without inhibiting other steroidogensis enzymes. The other aromatase inhibitors (steroidal androstenedione analogues) were irreversible in their effect on the aromatase enzyme and needed to be parentally administered. Most important, these old aromatase inhibitors were not potent enough to inhibit the aromatase and lower estrogen levels in women of the reproductive age group. A new group of non-steroidal aromatase inhibitors (letrozole, anastrazole and vorazole) is very potent and specific, reversibly inhibiting aromatase when orally administered with very high safety profile. Moreover, they have a relatively short half-life.
In Vitro Fertilization and In Vitro Maturation
It is useful to review in vitro fertilization and in vitro maturation for purposes of understanding the present invention. In vitro fertilization (IVF) in conventional use involves daily injections of fertility drugs, usually gonadotropins. Oocytes grow inside follicles and mature in the body. The mature oocytes are retrieved and fertilized by adding sperm in vitro in the laboratory.
In vitro maturation (IVM) does not necessarily involve the use of fertility drugs or may involve decreased doses of gonadotrophins when compared with IVF or OI. In the procedure, which is well documented in the literature, immature oocytes are retrieved from the female ovary at about day 7 for natural or artificial exogenous (gonadotrophins used) cycle and matured in vitro in the laboratory. Mature oocytes are then fertilized in the laboratory by intra-cytoplasmic sperm injection (ICSI). This procedure was developed for infertile women with polycystic ovary syndrome (PCOS). The advantages over in vitro fertilization (IVF) are reduced exposure to fertility drugs, reduced requirements for patient monitoring and because of the reduced exposure to fertility drugs, drug side effects are substantially eliminated, especially those associated with ovarian hyperstimulation syndrome (OHSS).
The procedure for IVM comprises the following steps:                Menstrual bleeding is induced by progestin.        Ultrasound (U/S) scan is performed on day 6-9 of the cycle.        Blood is drawn for maternal serum at time of U/S.        PCOS pattern of follicles without a dominant follicle (1.5 cm or greater) is seen at this time.        Human chorionic gonadotropin (hCG) 10,000 IU is given to the patient using vaginal ultrasound-guided needle aspiration and local anesthesia.        Egg retrieval 36 hours later and egg collection takes 15 to 30 minutes.        The harvested immature oocytes are cultured in maturation medium for 24 hours.        A fresh sperm sample from the female's partner is prepared for insemination.        The mature oocytes are then fertilized by ICSI.        Fertilization is checked at 16 hours after ICSI.        The fertilized oocytes are cultured for another 2 days.        Natural progesterone (Prometrium) is started vaginally (200 mg twice daily) on the day of oocyte insemination.        Two or three embryos (or one to two blastocysts) are transferred into the uterus.        Embryo transfer takes a few minutes and is painless.        